Variants of tryptophan synthases

Abstract

The present disclosure describes variant tryptophan synthases (TrpS) and their use in recombinant host cells to produce L-cysteine and / or L-cystine.

Description

Variants of tryptophan synthasesField

[0001] The present disclosure describes mutant tryptophan synthases (TrpS) and their use in recombinant host cells to produce L-cysteine and / or L-cystine.Background

[0002] Tryptophan synthase enzyme (TrpS) (EC 4.2.1.20) catalyzes the last step in the tryptophan biosynthetic pathway (Swift & Stewart, 1991). TrpS is composed of tpwo subunitsTrpA and TrpB which are organized in a heterotetrameric appa structure (Raboni, Bettati, & Mozzarelli, 2009) (Hyde, Ahmed, Padlan, Miles, & Davies, 1988). TrpS has received industrial attention because it is a promiscuous enzyme that can take various substrates and produce tryptophan analogs, S-substituted L-Cysteines, and non-canonical amino acids (Watkins-Dulaney, Straathof, & Arnold, 2021) (Wilcox, 1974) (Esaki, Tanaka, Miles, & Soda, 1983). It is commonly known that TrpS from E. coli can be used to produce cysteine via biocatalysis of serine and sodium hydrosulfide (NaSH) (Figure 1) (Ismail, Hashim, Jamal, Othman, & Salleh, 2014) (Ishiwata, Nakamura, Shimada, & Makiguchi, 1989). Pyridoxal 5'-phosphate (PLP) is required for the catalytic activity of TrpS because it serves as a co-factor. At high concentration and / or in presence of oxygen cysteine dimerizes to cystine as shown in Figure 1.Summary

[0003] In order to produce Cysteine and other analogues through biocatalysis economically, it is important to have more active TrpS in the biomass. Fraction of TrpS enzyme when overexpressed from strong promoters like T7 is known to fold incorrectly forming inclusion bodies. Furthermore, since Cysteine and other analogues are not natural substrate for TrpS, mutants are described herein providing improvements to the enzyme activity of TrpS.1. Accordingly, in a first aspect described herein is a mutant tryptophan synthase (TrpS) comprising two moieties, a and p, having amino acid sequences which are at least 70% identity to amino acid moieties SEQ. ID NO: 1 (TrpA) and 2 (TrpB) respectively, and comprising at least one mutation selected from: a) a substitution of an amino acid in a and / or ; b) a deletion of an amino acid in a and / or P; c) an insertion of an amino acid in a and / or P; and / or d) a fusion of a and P;compared to a parent TrpS comprising a and p moieties having amino acid sequences corresponding to SEQ ID NO: l and 2.

[0004] In a further aspect described herein is a polynucleotide gene encoding any of the mutant TrpS or a or or linker moieties described herein.

[0005] In a further aspect described herein is a polynucleotide construct comprising the gene described herein operably linked to a constitutive or inducive promoter.

[0006] In a further aspect described herein is a genetically modified host cell expressing or overexpressing the mutant TrpS or a or p or linker moieties described herein.

[0007] In a further aspect described herein is a cell culture, comprising the host cell described herein and a growth medium.

[0008] In a further aspect described herein is a method for producing cysteine or cystine comprising contacting L-serine with the TrpS mutant described herein in an aqueous medium in the presence of a source of sulfur to produce the cysteine or cystine; and optionally recovering and / or isolating cysteine or cystine.

[0009] In a further aspect described herein is a method for producing a biomass or catalytic inclusion body comprising the insoluble TrpS mutant fusion construct described herein comprising: a) propagating the cell culture described herein in a medium and at conditions allowing the cell to multiply and to produce the TrpS mutant fusion construct described herein; and b) optionally recovering and / or isolating biomass and / or catalytic inclusion body.Description of figures and referencesFigure 1: Production of L-cysteine via TrpS biocatalysis.Figure 2: Presumed mechanism of TrpS converting L-serine to L-cysteine and selected strategies for increasing specific enzyme activity.Figure 3: MutateX analysis to identify point mutations that stabilize the E(A-A) conformation more than the E(Aexl) conformation of TrpB. A Using MutateX, in silico saturation mutagenesis combined with stability predictions was conducted for TrpB E(A-A) (PDB 2J9X) and TrpB E(Aexl) (PDB 1KFJ). The TrpB E(Aexl) mutation AAG values were subtracted from the TrpB E(A-A) mutation_AAG values except if 1) the mutation AAG value of TrpB E(A-A) was > -1 2) the mutation AAG value of TrpB E(Aexl) was > 2 and 3) if the mutation AAG value of TrpB E(Aexl) < the mutation AAG value of TrpB E(A-A). In these three cases the mutation_AAG values were filtered away by setting the value to 0. These filtering choices were made in order to only select 1) mutations that significantly stabilizes the E(A-A) conformation of TrpB 2) mutations that does not significantly destabilize the E(Aexl) conformation of TrpB and 3) mutations that stabilizes the E(A-A) conformation more than the E(Aexl) conformation of TrpB. The limits of the subtracted_mutation_AAG values were set to -5 to 0 for proper visualization. B-l The subtracted_mutation_AAG values visualized in heatmaps for each residue in TrpB mutated to every amino acid. Each heatmap contains 50 residues of TrpB starting at residueT2 in B and ending at the final residue G395 in I.Figure 4: Design of TrpS variants with TrpA locked in its closed conformation. A Alignment of the open conformation (PDB 1KFJ) and the closed conformation of TrpA (PDB 2CLK) (RMSD of 0.352). B Disulfide bonds that lock TrpA in its closed conformation.Figure 5a: TrpS production plasmid (positive control plasmid).Figure 5b: Empty pSEVA_27 (negative control plasmid).Figure 5c: TrpAB fusion construct plasmid.Figure 6: Strain engineering workflow to construct E. coli strains expressing different TrpS variants.Figure 7: Primer design strategy for the mutation of one codon or multiple codons near each other.Figure 8: SDS-PAGE and BCA assay screening results for 12 TrpS solubility variants, the current TrpS production strain (positive control), and an identical strain with TrpS removed from the production plasmid (negative control). A SDS-PAGE of the soluble protein fraction (5 times diluted). B SDS-PAGE of the insoluble protein fraction (5 times diluted). C BCA assay of the soluble protein fraction. D BCA assay of the insoluble protein fraction.Figure 9: SDS-PAGE and BCA assay screening results for 9 TrpS solubility variants, the current TrpS production strain (positive control), and an identical strain with TrpS removed from the production plasmid (negative control). A SDS-PAGE of the soluble protein fraction (5 times diluted). B SDS-PAGE of the insoluble protein fraction (5 times diluted). C BCA assay of the soluble protein fraction. D BCA assay of the insoluble protein fraction.Figure 10: SDS-PAGE and BCA assay screening results for the last 5 TrpS solubility variants, the current TrpS production strain (positive control), and an identical strain with TrpS removed from the production plasmid (negative control). A SDS-PAGE of the soluble protein fraction (5 times diluted). B SDS-PAGE of the insoluble protein fraction (5 times diluted). C BCA assay of the soluble protein fraction. D BCA assay of the insoluble protein fraction.Figure 11: Biocatalysis results using batch fermentation biomass for 2 TrpS variants (S163R, N246Y) designed to have an increased E(A-A) stability, the currently used TrpS enzyme at industrial scale (+ve), and no TrpS enzyme (-ve).Figure 12: Total productivity during 2 h of biocatalysis using laboratory scale fed-batch fermentation biomass from the 2 most promising TrpS variants (S163R and N246Y) designed to have an increased E(A-A) stability and the current TrpS production strain (positive control).Figure 13: Structural investigation of S163R in energy-minimized homology models of TrpB made using SWISS- MODEL. The marked distances / polar interactions (stippled lines) are 2.8 A - 3.4 A if nothing else is indicated. A The E(Aexl) conformation of TrpB with S163 was made using PDB 1KFJ as template. B The E(A-A) conformation of TrpB with S163 was made using PDB 2J9X as template. C The E(Aexl) conformation of TrpB with R163 was made using PDB 1KFJ as template. D The E(A-A) conformation of TrpB with R163 was made using PDB 2J9X as template. R163 in the COMM domain stabilizes the E(A-A) conformation of TrpB by making several polar interactions with residues in the long loop.Figure 14: Biocatalysis results using batch fermentation biomass for S178C F212C and R179C G189C designed to have TrpA locked in its closed conformation, the currently used TrpS enzyme at industrial scale (positive control, +ve), and no TrpS enzyme (negative control, -ve).Figure 15: Total productivity during 2 h of biocatalysis using laboratory scale fed-batch fermentation biomass from S178C F212C and R179C G189C designed to have TrpA locked in its closed conformation and the current TrpS production strain (positive control, +ve).Figure 16: Structural investigation of R179C G189C in an energy-minimized homology model of TrpA made using SWISS-MODEL. A The closed conformation of TrpA with R179 and G189 was made using PDB 2CLK as template. The marked polar interactions (stippled lines) are 2.7 A - 3.1 A. B The closed conformation of TrpA with C179 and C189 was made using PDB 2CLK as template. C179 and C189 form a disulfide bond in Ioop6 locking the closed conformation of TrpA.Figure 17: Cysteine production from fusion constructs with various linker lengths.Figure 18: SDS-PAGE analysis of fusion constructsFigure 19: SDS-PAGE analysis of fusion construct used in example 7. "Sol" refers to soluble fraction, and "In" refers to insoluble fraction.Figure 20: Biocatalysis results reusing the soluble and insoluble fraction of the currently used TrpS enzyme (Cys_39) and the TrpAB fusion construct (Cys_197) A Production rate in round 1. B Production rate in round 2. C Production rate in round 3.Figure 21: recovery rate (%) of the soluble and insoluble fraction of the currently used TrpS enzyme (Cys_39) and the TrpAB fusion construct (Cys_197) after 20 min of biocatalysis.Figure 22: Biocatalysis results reusing the biomass of the TrpAB fusion construct (Cys_197).Incorporation by reference

[0010] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.DetailsDefinitions

[0011] Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1- 5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. http: / / enzyme.expasy.org / .

[0012] The terms "heterologous" or "recombinant" or "genetically modified" and their grammaticalequivalents as used herein interchangeably refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0013] The term "operative biosynthetic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0014] The term "substrate" or "precursor", as used herein refers to any compound that can be converted into a different compound. For clarity, substrates and / or precursors include both compounds generated in situ by a enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.

[0015] Term "endogenous" or "native" as used herein refers to a gene or a polypepetide in a host cell which originates from the same host cell.

[0016] The term "deletion" as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.

[0017] The term "disruption" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.

[0018] The term "attenuation" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.

[0019] The term "substitution", as used herein refers to a replacement of one amino acid in a polypeptide with another amino acid.

[0020] The term "fusion", as used herein refers to the covalent binding of a first polypeptide moiety to a second polypeptide moiety, optionally via a peptide linker moiety.

[0021] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0022] The term "and / or" as used herein is intended to represent an inclusive "or". The wording Xand / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0023] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0024] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences. The term "% identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues - x 100 Length of alignment — total number of gaps in alignmentThe term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides- - - x 100Length of alignment — total number of gaps in alignmentThe protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:Cost to open gap: default= 5 for nucleotides / 11 for proteinsCost to extend gap: default = 2 for nucleotides / 1 for proteinsPenalty for nucleotide mismatch: default = -3Reward for nucleotide match: default= 1Expect value: default = 10Wordsize: default = 11 for nucleotides / 28 for megablast / 3 for proteins.Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.

[0025] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0026] The term "expression vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expressioncassettes for the integration of genes into a host cell as well as plasmids and / or chromosomes comprising such genes.

[0027] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present disclosure. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0028] The term "polynucleotide construct" or "nucleic acid construct" refers to a polynucleotide, either single- or double stranded, which is separated from its naturally occurring environment and / or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0029] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.

[0030] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.

[0031] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0032] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0033] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed disclosure or to imply that certain features are critical, essential, or even important to the structure or function of the itemed disclosure. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0034] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells of the disclosure. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and yeast extract.

[0035] The term "fermentation medium" as used herein refers to any medium in which a genetically modified host is producing or has produced L-serine. A fermentation medium may contain solid cellular material (broths) or may be partially or wholly free of solid cellular material.

[0036] The term "sources of sulfur" as used herein refers to any compound comprising sulfur in a form which the TrpS mutant can covalently bind to serine to form cysteine.

[0037] The term "corresponding to" as used herein about amino acid or nucleotide residues refers to any residue that are placed similarly in 3D space.

[0038] Cys_39 is the strain having the TrpS production plasmid pCys_58 (SEQ ID NO: 391) expressing the reference TrpB moiety (SEQ ID NO: 2) and the reference TrpA moiety (SEQ ID NO: 1) forming the reference TrpS moiety (SEQ ID NO: 3) which is the currently used enzyme to produce L-cysteine and / or L-cystine, and it is referred to as the positive control, posC, and +ve.

[0039] Cys_309 is the strain having the empty pSEVA_27 (SEQ ID NO: 392). This is referred to as the negative control, negC, and -ve.

[0040] Cys_197 is the strain having the TrpA-13aa-TrpB fusion construct expressing plasmid which is similar to pCys_125 (SEQ ID NO: 393).

[0041] Combinations of mutations in one TrpS enzyme are indicated using "+" or which are used interchangeably.

[0042] The native TrpA a moiety from E. coli (SEQ ID NO: 876) has A189, but in this patent, G189 appears because the in silica structural analysis was done using TrpA from Salmonella enterica subsp. enterica serovar Typhimurium (PDB ID: 2CLK).It is to be noted that the TrpA a moiety used in the constructs used in the examples is N terminally His tagged for easy purification of protein is required, However, the amino acid residue numbers correspond to the native TrpA a moiety (SEQ ID NO: 876).EmbodimentsMutant tryptophan synthase (TrpS)

[0043] In some embodiments the two moieties, a and p, has amino acid sequences which are at least 75%, such at least 80%, such at least 85%, such at least 90%, such at least 95%, such at least 99% identity, such as 100% identical to amino acid moieties SEQ ID NO: 1 (TrpA) and 2 (TrpB).

[0044] In some embodiments the mutant TrpS has increased specific activity and / or increased or reduced water solubility compared to the parent TrpS.

[0045] In some embodiments the mutation promotes a fold type II protein conformation and demotes a fold type I protein conformation.

[0046] In some embodiments the mutation increases stability of a E(A-A) conformation of the moiety by introducing mutations that increases interactions between:e) a COMM domain corresponding to residue 102-189 of SEQ ID NO: 2, and f) a long loop domain corresponding to residue 260-310 of SEQ ID NO: 2, thereby increasing the activity of the mutant TrpS compared to the unmutated TrpS.

[0047] The COMM domain of SEQ ID NO: 2 (TrpB) is:GKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRD WSGSYETAHYMLG

[0048] The long loop domain of SEQ ID NO: 2 (TrpB) is:HGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSISAGLDFPSVG

[0049] In some embodiments the mutation increases or locks a closed conformation of the a moiety by introducing mutation in a Loop6 domain of the a moiety corresponding to residue 178-192 of SEQ ID NO: 1, and thereby increases the activity of the mutant TrpS compared to the unmutated TrpS.

[0050] The Loop6 domain of SEQ ID NO: 1 (TrpA) is: SRAGVTGAENRAALP

[0051] In some embodiments the mutant TrpS comprises one or more substitutions in the moiety moiety selected from:A226E, V227P, V227D, C230P, C230E, V231D, V276P, V276K, V276E, G277D, I278K, I278E, I278D, Y279K, Y279E, F280E, Q370E, L371K, L372K, V374P, N375P, N375D, L376K, S377P, S377E, A226E+C230P, V276P+I278D, L371K+N375P+S377P, A226E+V227P+C230P+V231D,V276P+G277D+I278D+Y279E+F280E, Q370E+L371K+L372K+V374P+N375P+L376K+S377P, T190F, Q114Y, E256K, D329K, A24P, N246Y, D329R, Q114F, D329M, T190L, S163R, D329T, D329F, D329Q, A242M, D329A, E256T, H388F, D329E, D329I, E256P, S161R, S163H, D329V, S163F, E256H, S163Y, R379Y, D329Y, E295H, S163L, A112M, K167R, A31I, S34L, S178L, H388L, H388W, E296W, D247Y, T190E, S104P, H357Y, F45Y, T190M, S163K, H388M, D247H, S34F, H357F, S34K, Q114V, E334F, L166Y, E334W, E203Q, Q114K, Q27Y, T199H, E334M, L166F, S34Y, T190D, S161Y, D247Q, H185F, E256M, R379L, D247F, H115S, S34R, S34W, E296F, D247K, D247L, H115C, D243H, T386M, D243P, E296Y, Q205E, S34M, E296Q, D247R, Q114I, C230P, A85S, R321M, D247M, S180N, D330W, R150Y, R379C, H185Y, D247I, R379T, D243C, A302P, D243S, D247V, K50W, R379M, E203M, R202M, E256L, Q27F, Q114M, E296M, H115A, E295A, T386F, D243R, E296N, K50F, E367F, E256V, E220R, H185M, T199F, S301C, Q205S, Q36Y, H86A, E367Y, Q27M, T183Q, E220Y, R379G, E256A, S161P, T386P, R363W, E256C, S318M, N246P, S249I, E256N, E295W, T2G, R379S, D168P, E343K, Q114E, S161W, E364M, T183L, E11M, S326Y, T199Y, D330P, D243F, N26W, E210R, A393R, H185L, A112P, D243K, T338L, L125M, K50L, T199V, E367W, D243I, E220M, E343L, S318L, S143M, C230A, T199L, E105W, H86E, S104Q, Q42W, D243M, D243L, H160W, R100Y, S249R, T183M, T386L, T72I, D243A, E266R, S249K, S104M, Q27L, E364K, E210I, D323W, I300M, R379Q, T72N, D218T, D323M, Q215F, E295Y, E105M, T386Y, 1245L, E30K, E296L, E369P, E296I, V139M, E343M, V139I, D243Y, E210L, A184V, K213R, S249M,K129Y, A46N, S249L, T386W, N26F, E296K, S143L, D243W, E295K, S161L, E220F, E295M, E266F, E343C, Q63P, D330M, E364Q, Q114L, E266Y, A214M, S143Y, R379K, T199M, E295F, K283R, C340A, E369R, E295L, Q36L, S326E, A393K, D323V, D47W, H260L, E266L, K283L, T328S, E30R, D243Q, H273M, Q205L, S318Q, S143R, E105L, H160L, D243E, E296R, D243V, E343Q, T338I, A214K, T183R, 1262 P, T199I, E364R, H260M, H357L, Q44M, K283M, E210M, E210K, K103R, V156M, I238M, S326K, H357M, A393H, A214I, 81041, H160M, V276L, D218S, N26L, Q365R, H115L, R321L, K129M, K337L, A393S, H160P, L333M, S104L, E266K, D323Q, E155L, D218M, Q19P, D323E, E30Y, S318K, D218N, H273F, A290P, S104K, E210Q, H357W, D218A, S326R, H260Q, A290E, Y315W, T183I, S104R, R379F, T2R, E109H, E109D, L166K, L166H, L166Q, C170N, A302G, F306H, E109D+L166Q+C170N+A302G+F306H.

[0052] In some embodiments the mutant TrpS comprises one or more substitutions in the p moiety selected from: S163R, N246Y

[0053] In some embodiments the mutant TrpS comprises one or more substitutions in the a moiety selected from: Y4C+G172C, S178C+F212C, R179C+G189C, A185C+S215C, A185C+S235C, Y4C+G172C+A185C+S235C, R179C+G189C +A185C+S235C.

[0054] In some embodiments the mutant TrpS comprises one or more substitutions in moiety a selected from: S178C_F212C, R179C_G189C

[0055] In some embodiments the moiety comprises an amino acid sequence as set forth in anyone of SEQ ID NO: 11 to 366.

[0056] In some embodiments the a moiety comprises an amino acid sequence of the as set forth in anyone of SEQ ID NO: 4 to 10.

[0057] In some embodiments the a and p moieties are fused into a fusion construct.

[0058] In some embodiments the fusion construct comprises a linker sequence between a and p moieties.

[0059] In some embodiments the linker sequence has a sequence which is at least 70%, such as at least 75%, such at least 80%, such at least 85%, such at least 90%, such at least 95%, such at least 99%, such as 100% identical to the linker comprised in SEQ ID NO: 379 to 390.

[0060] In some embodiments the fusion construct has an amino acid sequence which is at least 70%, such as at least 75%, such at least 80%, such at least 85%, such at least 90%, such at least 95%, such at least 99%, such as 100% identical to the fusion construct comprised in anyone of SEQ ID NO: 367 to 378.

[0061] In some embodiments the fusion construct is less soluble in aqueous solution than the unfused a and p moieties.

[0062] In some embodiments the fusion construct is insoluble in aqueous solution.

[0063] In some embodiments the fusion construct remains catalytically active in aqueous solutioneven when the fusion construct is less soluble or insoluble in aqueous solution. It was a significant surprise that even when the fusion construct became insoluble, it remained catalytically active and this property represents a significant advantage for the use of biomasses comprising the insoluble fusion construct such as in a repeated use of enzyme by centrifugation and / or immobilization of enzyme or biomass.

[0064] In some embodiments the mutant TrpS is a heterotetramer comprising appa in a heterotetrameric structure.

[0065] The present invention also relate to polynucleotide genes encoding any of the mutant TrpS or a or p or linker moieties described herein, and polynucleotide constructs comprising the gene operably linked to a constitutive or inducive promoter.

[0066] In some embodiments the polynucleotide construct is an expression vector.The present invention also relate to a genetically modified host cell expressing or overexpressing the mutant TrpS or a or p or linker moieties described herein.

[0067] In some embodiments the host cell expresses one or more further enzymes in a pathway producing Cysteine or cystine.

[0068] In some embodiments the host cell is a prokaryotic cells, optionally a bacterium.

[0069] In some embodiments the prokaryotic cell is a cell of Pseudomonadota, optionally of the class gammaproteobacteria, optionally of the family Enterobacteriaceae, optionally of the genus Escherichia, optionally of the species Escherichia coli.

[0070] In some embodiments the the prokaryotic cell is an Actinomycetota, optionally of the class Actinobacteria, optionally of the family Corynebacteriaceae, optionally of the genus Corynebacterium, optionally of the species Corynebacterium glutamicum.

[0071] In othe embodiements the bacteria which can be used according to the present disclosure belong to the Enterobacteriaceae family, such as bacteria belonging to a genus selected from the group consisting of Escherichia, Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia, Ewingella, Gibbsiella, Hafnia, Klebsiella, Leclercia, Leminorella, Lonsdalea, Mangrovibacter, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Phaseolibacter, Photorhabdus, Plesiomonas, Proteus, Rahnella, Raoultella, Saccharobacter, Salmonella, Samsonia, Serratia, Shimwellia, Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Yersinia and Yokenella.

[0072] According to certain other embodiments, the bacterium belongs to a genus selected from the group selected from Escherichia, Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Streptococcus, Pseudomonas, Streptomyces, Shigella, Acinetobacter, Citrobacter,Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus and Yersinia.

[0073] According to particular embodiments, the bacterium belongs to the genus Escherichia. According to particular embodiments, the bacterium is Escherichia coli. Non-limiting examples of a bacterium belonging to the genus Escherichia, which can be used to derive a host cell of the present disclosure are Escherichia coli K-12 (especially substrain MG1655 or W3110), BL21, W, or Crooks. According to more particular embodiments, the bacterium is Escherichia coli K-12.

[0074] According to other particular embodiments, the bacterium belongs to the genus Corynebacterium. A non-limiting example of a bacterium of the genus Corynebacterium is Corynebacterium glutamicum. According to other particular embodiments, the bacterium is Corynebacterium glutamicum.

[0075] According to other particular embodiments, the bacterium belongs to the genus Bacillus. Nonlimiting examples of a bacterium of the genus Bacillus are Bacillus subtitlis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus mojavensis. According to more particular embodiments, the bacterium is Bacillus subtitlis. According to other particular embodiments, the bacterium is Bacillus licheniformis.

[0076] According to other particular embodiments, the bacterium belongs to the genus Lactococcus. A non-limiting example of a bacterium of the genus Lactococcus is Lactococcus lactis. According to other particular embodiments, the bacterium is Lactococcus lactis.

[0077] According to other particular embodiments, the bacterium belongs to the genus Streptomyces. A non-limiting examples of a bacterium of the genus Streptomyces are Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus. According to other particular embodiments, the bacterium is Streptomyces lividans. According to other particular embodiments, the bacterium is Streptomyces coelicolor. According to other particular embodiments, the bacterium is Streptomyces griseus.

[0078] According to other particular embodiments, the bacterium belongs to the genus Pseudomonas. A non-limiting example of a bacterium of the genus Pseudomonas is Pseudomonas putida. According to more particular embodiments, the bacterium is Pseudomonas putida.Methods

[0079] The present invention also relates to a method for producing cysteine or cystine comprising contacting L-serine with the TrpS mutant in an aqueous medium in the presence of a source of sulfur to produce the cysteine or cystine; and optionally recovering and / or isolating cysteine or cystine.

[0080] In some embodiments the medium further comprises ammonium ions, optionally between 0,1g / L to 20 g / L, optionally between 0,5 g / L to 15 g / L, optionally between 1 g / L to 10 g / L, optionally between 2 g / L to 5 g / L of ammonium ions.

[0081] In some embodiments the medium further comprises phosphate ions, optionally between 0,01 g / L to 10 g / L, optionally between 0,02 g / L to 5 g / L, optionally between 0,05 g / L to 2 g / L of phosphate ions.

[0082] In some embodiments the medium further comprises at least 0,1 pM of pyridoxal 5-phosphate (PLP cofactor). The medium preferably comprises pyridoxal 5-phosphate (PLP) cofactor in amounts of at least 0,01 pM, such as at least 0,05 pM, such as at least 0,1 pM, such as at least 0,5 pM, such as at least 1 pM, such as at least 5 pM, such as at least 10 pM, such as at least 50 pM, such as at least 100 pM, such as at least 250 pM, such as at least 500 pM, such as at least 1 mM but suitably not higher than 10 mM.

[0083] In some embodiments the medium comprises at least 10 g / L of serine, such as between 50 to 500 g / L by weight of serine, optionally prior to conversion of any serine into cysteine and / or cystine.

[0084] In some embodiments the source of sulfur is capable of forming HS_ions, optionally selected from HjS, S2-, and NaHS.

[0085] In some embodiments the source of sulfur comprises hydrogen sulfide.

[0086] In some embodiments the source of sulfur comprises basic compounds, optionally strongly basic or comprises acidic compounds, optionally strongly acidic.

[0087] In some embodiments the medium comprises a concentration of at least 0,1 % wt of the source of sulfur.

[0088] In some embodiments the concentration of at least 0,1 % wt of the source of sulfur in the medium is maintained during the conversion of serine to cysteine.

[0089] In some embodiments the method comprises: a) culturing the cell culture of disclosed herein in a medium and at conditions allowing the cell to produce cysteine or cystine; and b) optionally recovering and / or isolating cysteine or cystine.

[0090] In some embodiments the methodfurther comprises one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.

[0091] In some embodiments themethod comprises feeding the cell culture exogenously with one ormore cysteine or cystine precursors.

[0092] In some embodiments the the recovery and / or isolation step comprises to one or more steps selected from: a) contacting the medium with one or more adsorbent resins to obtain at least a portion of the produced Cysteine or cystine; b) contacting the medium with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the Cysteine or cystine; c) extracting the Cysteine or cystine; and / or d) precipitating the Cysteine or cystine by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the Cysteine or cystine by filtration or gravity separation; thereby recovering and / or isolating the Cysteine or cystine.

[0093] The invention also relates to a method for producing a biomass or catalytic inclusion body comprising the insoluble TrpS mutant fusion construct disclosed herein comprising: g) propagating the cell culture disclosed herein in a medium and at conditions allowing the cell to multiply and to produce the TrpS mutant fusion construct disclosed herein; and h) optionally recovering and / or isolating biomass and / or catalytic inclusion body.Biomasses or preparations there of comprising the insoluble, yet catalytically active, mutant TrpS fusion construct and be added to solutions comprising Serine and the source of sulfur for conversion into cysteine or cystine and this biomass or preparation thereof can be reused multiple times.SequencesThe present application embodies the sequences of the following table:Examples Materials and methods

[0094] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade.Introduction

[0095] To improve the amount of active TrpS we attempted to increase the solubility and activity of the enzyme. All the bioinformatic tools used are given in example 1. The key mutations identified from the computational analysis were introduced in the plasmid pCys_58 (SEQ ID NO: 391). All the molecular biology details are given in example 2. The obtained strains were tested for biocatalysis for production of L-cysteine. The details of the methods are given in example 3 while the screening and characterization results are given in example 4. Furthermore, we were interested to have more stable version of TrpS which can be used multiple times for biocatalysis which will significantly reduce the production cost of the products. Therefore, we fused TrpA and TrpB units using various linkers and toour surprise the fusion constructs were completely insoluble but highly active. This has never been reported before for TrpS. These novel fusion constructs have led to become catalytically active inclusion bodies (catIB). We demonstrate the construction of these fusion constructs in example 5, the study of various fusion constructs in example 6, and the repeated use of a TrpS fusion construct in more than one biocatalysis cycle in example 7.Example 1: Computational analysis to rationally engineer TrpS l.l.ConSurf

[0096] The ConSurf webserver was accessed through this link: http: / / consurf.tau.ac.il (Ashkenazy, et al., 2016). It has been used to analyze the conservation of each residue in TrpB from E. coli (PDB 2DH5) using the default settings of ConSurf. The conservation of residues is calculated based on the sampling of homologous sequences which is used to create a multiple-sequence alignment for the construction of a phylogenetic tree. Position-specific evolutionary rates are calculated and normalized to output the ConSurf Grade. The least conserved residues are graded 1, and the most conserved residues are graded 9. In addition to the ConSurf grade, the residue variety in % for each position is calculated.1.2 PDB structures

[0097] PyMOL has been used to visualize and modify PDB structures for the needs of this study. The text editor software, SublimeText, has been used to write PyMOL scripts. Alignment of PDB structures was conducted using the 'align' or the 'super' function in PyMOL. 'align' was used if the structures have a high sequence identity, 'super' was used if the sequence identity of the two structures is low. For the visualization of PDB structures, they were rendered at ray trace mode 1, antialias was set to mode 4, and the background was set to white. The fully automated protein structure homologymodelling server SWISS-MODEL has been used to generate energy-minimized PDB structures to investigate the effect of mutations. A target sequence, either TrpA or TrpB, was submitted and a proper TrpS PDB structure was used as template for building the homology model.1.3 Solubis

[0098] The Solubis webserver (http: / / solubis.switchlab.org / ) has been used to design TrpS solubility variants (Van Durme, et al., 2016). Before submission of TrpB from E. coli (PDB 2DH5), PLP and glycerol were removed from the PDB file using PyMOL because Solubis cannot recognize the molecules. Solubis' standard settings were used: 1) repair PDB using FoldX Repair PDB 2) consider chain A (this is TrpB) 3) TANGO threshold of 5 4) mutate each residue to all gate-keeper residues (Asp, Glu, Arg, Lys, Pro) which is referred to as APR flanking mutations. The output is a result folder. The solubis resultstab file (data converted to be opened in excel) contains all the calculated TANGO and free energy values.

[0099] One way to increase the solubility and thereby the expression of functional TrpB is by decreasing its aggregation tendency. Solubis is an easy-to-use webserver that only requires the submission of a PDB file to predict aggregation prone regions (APRs) and mutations within the APRs potentially suppressing aggregation while taking into account thermodynamic stability (Van Durme, et al., 2016). Solubis uses the TANGO algorithm and the empirical force field FoldX to calculate protein aggregation and thermodynamic stability, respectively (Schymkowitz, et al., 2005) (Fernandez- Escamilla, Rousseau, Schymkowitz, & Serrano, 2004). E. coli TrpB (PDB 2DH5) was analyzed using Solubis, and it turned out to have 3 APRs. APR3 (residue 370-377) has the highest summed TANGO score which is approximately 300 compared to APR1 (residue 226-231) and APR2 (residue 276-280) which both have a summed TANGO score of about 90, suggesting that APR3 has the highest aggregation propensity. The effect of a mutation on protein aggregation is evaluated based on ATANGO which is calculated by subtracting TANGOWii tyPe from TANGOmutant- If ATANGO is negative, the mutation decreases the aggregation propensity of the protein and vice versa. The summed AG of an APR conveys its stability. The effect of a mutation on protein stability is evaluated based on AAG which is calculated by subtracting AGWiid type from AGmutant. It is a destabilizing mutation if AAG is positive, and it is a stabilizing mutation if AAG is negative.

[0100] APR flanking mutations were investigated for each residue in all the APRs resulting in a list of 95 mutations (30 for APR1, 25 for APR2, and 40 for APR3). 89 out of the 95 mutations were of interest because they have a negative ATANGO score, hence decreasing aggregation propensity of TrpB.

[0101] However, also evaluating the mutations based on their effect on protein stability, we decided to engineer and screen 31 TrpS variants. The key substitutions by the Solubis analysis are mentioned in Table 1. The TrpB Mutants have SEQ ID NO: 11 to 41.incorporated in TrpB. ATANGO: a negative score means that the mutation decreases aggregation, and a lower score decreases aggregation the most. AAG (kcal / mol): a negative score means that the mutation is stabilizing, and a lower score is more stabilizing. A positive score means that the mutation is destabilizing, and a higher score is more destabilizing. ConSurf Grade: the conservation of the original residue, 1 is not conserved, and 9 is highly conserved. Location: the structural location of the residue to mutate.1.4 MutateX

[0102] MutateX is an automated pipeline for in silico saturation mutagenesis of protein structures (Tiberti, et al., 2022). The Python code for running MutateX is available on GitHub: https: / / github.com / ELELAB / mutatex. MutateX applies FoldX to calculate the free energy AG and the AAG (in kcal / mol) (Schymkowitz, et al., 2005):

[0103] We used a novel MutateX design approach to identify if any mutations stabilize the E(A-A) conformation more than the E(Aexl) conformation of TrpB. This has been done by separately performing in silico saturation mutagenesis of TrpB in the E(Aexl) and the E(A-A) conformation. It is of interest to further stabilize the E(A-A) conformation of TrpB because this is the catalytically competent conformation. Stabilizing the E(A-A) conformation to lower its Gibbs free energy and hence shift the equilibrium between E(Aexl) and E(A-A) towards E(A-A) will result in an increased accumulation of the active E(A-A) conformation of TrpB as shown in Figure 2 (strategy 1).

[0104] TrpB E(Aexl) (PDB 1KFJ) was used while we chose to use PDB 2J9X as the TrpB E(A-A) conformation. MutateX was set to perform saturated mutagenesis of the p-subunits in the given biologically active heterotetramer mutating the same residue in both -subunits simultaneously. The output is a data file for each mutated residue pair containing the calculated mutation_AAGs (kcal / mol) for each mutation. The result of the MutateX analysis is shown in Figure 3. As we were only interested in the mutations that stabilize the E(A-A) the most without significantly destabilizing the E(Aexl), the data was filtered as explained in Figure 3a. After filtering, the AAGs of the E(Aexl) was subtracted from the AAGs of the E(A-A) to identify mutations that stabilize the E(A-A) more than the E(Aexl). E(A-A) is more stabilized by the mutation than the E(Aexl) if the subtracted_mutation_AAG < 0. All the subtracted_mutation_AAGs for each residue in TrpB (x-axis) mutated to all the natural amino acids (y- axis) are visualized in heatmaps (Figure 3B-I). Several mutations (dark spots) appear to stabilize the E(A-A) conformation more than the E(Aexl) conformation. These mutations are listed in Table 2, and the TrpB Mutants have SEQ. ID NO: 42 to 357.| T2R | -1.44288 | -1.43600-0.00688Table 2: Mutations that stabilize the E(A-A) conformation more than the E(Aexl) conformation of TrpB (based on the MutateX analysis shown in Figure 3). AAG (kcal / mol): a negative score means that the mutation is stabilizing, and a lower score is more stabilizing. A positive score means that the mutation is destabilizing, and a higher score is more destabilizing.1.5: disulfide bond engineering using PyMOL

[0105] Strategy 2 (visualized in Figure 2) is to lock TrpA in its closed conformation to ensure constant signalling to TrpB to adopt its closed conformation E(A-A). Considering it is the binding of IGP that induces the closed conformation of TrpA and no IGP is present during for example cysteine biocatalysis, we do not expect TrpA to populate the closed conformation. Since TrpA does not populate its closed conformation, it does not signal TrpB to populate its closed E(A-A) conformation. The lack of signaling from TrpA to TrpB can make the conformational change of TrpB even more ratelimiting than it naturally is. Strategy 2 is a way to reintroduce signalling from TrpA to TrpB, and indirectly stabilize the closed E(A-A) conformation of TrpB. It should be noted that this is only possible because the catalytic activity of TrpA is not necessary for e.g. cysteine production.

[0106] Using PyMOL, the open conformation of TrpA was extracted from PDB 1KFJ, and the closed conformation of TrpA was extracted from PDB 2CLK. The open and closed conformation of TrpA was structurally aligned having an RMSD of 0.352 (Figure 4a). Loop6 in the closed conformation of TrpA is especially noticeable because it is not modelled in the open conformation of TrpA, likely due to its high flexibility resulting in poor electron density (Figure 4a). Loop6 in the closed conformation of TrpA is an obvious target for disulfide engineering to lock the closed conformation (Figure 4b). 7 TrpA variants (Y4C_G172C, S178C_F212C, R179C_G189C, A185C_S215C, A185C_S235C,Y4C_G172C_A185C_S235C, R179C_G189C _A185C_S235C) were designed, and the TrpA Mutants have SEQ ID NO: 4-10.1.6 PocketOptimizer

[0107] Strategy 3 (visualized in Figure 2) is packing of the active site of the closed conformation of TrpB E(A-A) around for example SH- to optimize the binding and positioning of SH-, thereby improving the nucleophilic attack to produce for example cysteine. The active site is evolved to fit indole which is a much bigger molecule than hydrosulfide. There is a reason to believe that the current addition of hydrosulfide to the E(A-A) intermediate is not optimal, and it can be improved by fitting the active site around hydrosulfide.

[0108] PocketOptimizer is a modular framework for computer-aided ligand-binding design (Noske, Kynast, Lemm, Schmidt, & Hocker, 2023). The Python code for running PocketOptimizer 2.0 is available on GitHub: https: / / github.com / Hoecker-Lab / pocketoptimizer. It was run through Jupyter Notebook. A PDB file and a ligand file are required to run PocketOptimizer. The E(A-A) conformation of TrpB was extracted from PDB 2J9X and the crystal content was removed from the PDB using PyMOL. Hydrogen sulfide (SHz) was downloaded from RCSB PDB (ID: H2S) as an SDF file and used as the ligand file. Before running PocketOptimizer, the E(A-A) (PDB 2J9X) and the E(Aexz) (PDB 6AM8) conformation of TrpB were aligned in PyMOL, and then hydrogen sulfide was superpositioned on the indole carbon making the nucleophilic attack using the Pair Fitting function. The aligned E(A-A) conformation of TrpB and hydrogen sulfide was used in the PocketOptimizer analysis. The default settings were used, pH was set to 9, and one residue was mutated at a time to all amino acids except the original one. PocketOptimizer calculates packing and binding energies using the AMBER ffl4SB force field, and 50 structures (including different rotamers) with the lowest energy were summarized in an energy report. They key mutations from this analysis are mentioned in Table 3, and the TrpB Mutants have SEQ ID NO: 358 to 366.Table 3: 9 TrpS variants were designed to have a TrpB active site optimized for the hydrosulfide substrate (SH ). ConSurf Grade: the conservation of the original residue, 1 is not conserved, and 9 is highly conserved. Binding energy: a negative value indicates that the binding affinity to hydrosulfide is increased. Packing energy: a negative value indicates that the stability of the protein is increased. Total energy: the sum of the packing energy and the binding energy.Example 2: Strain Engineering2.1 Plasmid mapsThe key plasmids used are shown as plasmid maps in Figure 5. pCys_58 (SEQ ID NO: 391) is the currently used TrpS production plasmid, and it has been used to express TrpS as positive control(Figure 5a). The empty pSEVA_27 (SEQ ID NO: 392), which does not express TrpS, has been used as negative control (Figure 5b). pCys_125 (SEQ ID NO 393) is a TrpAB fusion construct expressing plasmid (Figure 5c).2.2 Site directed mutagenesis for introduction point mutations

[0109] The workflow followed for the construction of E. coli strains expressing different TrpS variants is shown in Figure 6. Previously described plasmid pCys_58 contained D47S mutation in TrpB was used as the template for these PCR. This mutation has been identified to be beneficial for TrpB activity in previous studies and incorporate mutations in the gene encoding TrpS. The PCR product was then transformed into E. coli NEB5a (cloning strain) to repair the nick of the TrpS plasmid. Sanger sequencing was used to confirm that the mutation(s) were incorporated correctly in the gene encoding TrpS. If this was the case, the TrpS plasmid was subsequently transformed into E. coli BL21(DE3) (production strain).

[0110] The primer design strategy and the PCR program are shown in Figure 7 and Table 3, respectively. The primers used to construct TrpS solubility variants are listed in Table 4. The primers used to construct TrpS variants with stabilized E(A-A) confirmation of TrpB are listed in Table 5. The primers used to construct TrpS variants with TrpA locked in its closed conformation are listed in Table 6.AB CTable 3: PCR protocol. A PCR reaction mixture. B Touchdown PCR program. C Gradient PCR programTable 4: Primers used to construct TrpS solubility variantsTable 5: Primers used to construct TrpS variants with stabilized E(A-A) confirmation of TrpBTable 6: Primers used to construct TrpS variants with TrpA locked in its closed conformation.2.3 Gibson assembly for introduction multiple mutations

[0111] Gibson assembly was used to construct the TrpS variants with disulfide bonds in TrpA because it required mutating two residues to cysteine that were not sequentially close to each other. The current TrpS production plasmid was used as template for amplifying both the insert and the vector.Touchdown PCR was conducted, and then the PCR products were Dpnl digested and purified. The NEBuilder Hifi DNA Assembly Reaction Protocol was followed to do the Gibson assembly. The total amount of DNA (insert +vector) in the reaction mixture was 0.15 pmol. If the insert was less than 200 bp, it was added in 5-fold molar excess compared to the vector. If the insert was more than 200 bp, it was added in 2-fold molar excess compared to the vector. The following equation was used to calculate the amount of DNA: ng = (pmol * bp * 650daltons) / 1000. 5pL NEBuilder HiFi DNA Assembly Master Mix (product number: E2621S) and ddHzO were added to the reaction mixture to reach a total volume of 10 pL. Samples were incubated at 50°C for 1 h. NEB5a was transformed with 5 pL of theassembled product.2.4 Transformation of chemically competent E. coli

[0112] If needed, chemically competent cells were made using Transformation and Storage Solution (TSS). A pre-culture was started in 3 mL LB media with no or appropriate antibiotics and incubated at 37°C 250 RPM overnight (Eppendorf Innova S44i incubator). In a 250 mL shake flask, 0.5 mL preculture was inoculated in 50 mL LB media with no or appropriate antibiotics (100-fold dilution). The fresh culture was incubated at 37°C 250 RPM (Eppendorf Innova S44i incubator) for approximately 2.5 h until OD600 reached 0.2-0.5. The shake flask with freshly grown cells was placed in ice for approximately 15 min and when cooled down, the culture was transferred to a 50 mL falcon tube which was centrifuged at 4°C 4000 x g for 5 min. The supernatant was discarded and the 50 mL falcon tube with the cell pellet was placed on ice. The cell pellet was gently resuspended in 2.5 mL ice-cold TSS-buffer (5% of original culture volume) by pipetting. 50 pL chemically competent cells were distributed to precooled 1.5 mL sterile eppendorf tubes or a precooled sterile PCR plate (sterilized at 110°C for 5 min and then cooled down on ice). The cells were transformed immediately or stored at - 80°C. No glycerol was added because it is already a part of the TSS-buffer 5 pL PCR product or 1-2 pL plasmid was added to thawed 50 pL chemically competent cells on ice. After approximately 30 min. on ice, the cells were heat shocked at 42°C for 45 sec. using a water bath or a heating block. If transforming in eppendorf tubes, 950 pL SOC media was added, and the cells were recovered in a heating block for approximately 1 h at 37°C 800 RPM (Eppendorf ThermoMixer C). The cells were spun down at 5000 x g for 2 min, and most of the supernatant was discarded. The cells were resuspended in the 50-100 pL leftover supernatant, plated on an LB agar plate with the appropriate antibiotic, and incubated overnight at 37°C. If transforming in a PCR plate, the cells were transferred to a squared 96- deep well plate with 300 pL SOC media. The cells were recovered for approximately 2 h at 37°C 250 RPM (Eppendorf Innova S44i incubator). All the volume (50 pL cells + 300 pL SOC media) was plated on an LB agar plate with no or appropriate antibiotic using beats to spread out, and the plate was dried in the LAF bench for approximately 10 min. and then moved to a 37°C incubator overnight.2.5 Sanger sequencing

[0113] Constructed TrpS plasmids were isolated from E. coli NEB5a using a Nucleospin plasmid miniprep kit from MACHEREY-NAGEL, and the isolation of high-copy plasmid DNA protocol was followed. The constructed TrpS plasmids were sent for Sanger sequencing to confirm that the mutation(s) were incorporated correctly. ~5 pL purified plasmid, 2.5 pL 10 pM primer, and ~2.5 pL ddl-120 were mixed and sent for sequencing at Eurofins genomics using their Mix2Seq kit.Example 3: Screening methods3.1 Autoinduction in shake flasksTo harvest biomass for TrpS screening, strains were grown in shake flasks in autoinduction media to express TrpS. The composition of the autoinduction media can be seen in Table 7. On the first day, 5 mL 2xYT+0.25% glucose+Kan50 cultures in 50 mL falcon tubes were started from cryostocks in the morning and incubated at 37°C 250 RPM for ~9 h (Eppendorf Innova S44i incubator). 250 mL shake flasks with 25 mL autoinduction media were prepared and inoculated with a culture volume resulting in OD600 0.05. For the OD600 measurements, the overday cultures were diluted 25 times. The inoculated shake flasks were incubated at 37°C 250 RPM for 2 h and hereafter at 28°C 250 RPM for 10 h to secure conditions for TrpS to fold (Eppendorf Innova S44i incubator). The total incubation time for the expression of TrpS was 12 h. The 25 mL cultures were poured into pre-cooled 50 mL falcon tubes placed on ice. The tubes were weighed beforehand. A 40 times dilution was used to measure OD600 of the autoinduced cultures. OD600 of the 12 h autoinduced shake flask cultures was ~15. Triplicates of 2 OD units (2 / OD600) of each culture were distributed to pre-cooled eppendorf tubes placed on ice, and they were centrifuged at 16000 x g 4°C for 2 min. The supernatant was discarded by pipetting, and the biomass was stored at -20°C until used for screening. The rest of the culture broth in the cold 50 mL falcon tubes was centrifuged at 10000 x g 4°C for 10 min. The supernatant was discarded by pouring, and the falcon tubes were shortly placed upside-down on a paper towel to remove the last supernatant. The falcon tubes with biomass were placed on ice and weighed before storing at -20°C to note down the exact g wet weight of cells in each falcon tube.Table 7: composition of autoinduction media3.2 Laboratory scale fermentationSelected TrpS strains were grown in fed-batch fermentation (1 L working volume) to harvest the biomass for biocatalysis. First, bullets (single-use glycerol stocks) were prepared by streaking theselected TrpS strains from cryostocks on LB agar plates with kanamycin. After overnight incubation at 37°C, 10 mL 2xYT+0.25% glucose+Kan50 cultures were made in 50 mL falcon tubes and incubated at 37°C 250 RPM for 6-8 h (Eppendorf Innova S44i incubator). The OD of the cultures was measured and reported in a bullet inventory. 3-6 bullets (1 mL 10% glycerol stocks) were made for each TrpS strain and stored at -80°C. On the day of the fermentation, one bullet of each strain was thawed and 250 pL was inoculated in 25 mL 2xYT+0.25% glucose+2 mM MgSO4+Kan50 in 250 mL shake flasks. The seed cultures were incubated at 37°C 250 RPM for 6-8 h (Eppendorf Innova S44i incubator). The fed-batch fermentation had 2.5% seed culture inoculum to a start. The composition of the batch and feed media is summarized in Table 8, and 50 pg / mL kanamycin was added to all fermentors. The feed was started after the depletion of batch glucose (after ~6 h) and 400 pM IPTG induction was triggered by target OD600 ~30 (after ~15 h). The pH was kept at 7 using 15% NH3 for regulation. The initial temperature was 37°C, and it was lowered to 28°C after IPTG induction. The temperature regulation happened over 1 h. The fermentation was stopped after 22 h, and the fermentation process was consistent for all strains. The fermentation broth was kept cold at all times when sampling. 2 OD units (2 / OD600) of biomass for SDS-PAGE were sampled in eppendorf tubes 'pre-induction' (1-2 h after inoculation) and 'post-induction' (end of fermentation) by centrifugation for 2 min. at 16000 x g 4°C. The biomass was stored at -20°C. At the end of the fermentation, the broth from each fermentor was poured into precooled 1 L bluecap flasks and stored at 4°C during sampling. From each bluecap flask, 40 mL was added to 6 cold 50 mL falcon tubes (weighed beforehand) which were then centrifuged for 10 min. at 10000 x g 4°C. The supernatant was discarded by pouring, and the falcon tubes were shortly placed upsidedown on a paper towel to remove the last supernatant. The falcon tubes with biomass were placed on ice and weighed before storing at -80°C to note down the exact g wet weight of cells in each falcon tube.Table 8: composition of laboratory scale fed-batch fermentation media3.3 SDS-PAGE

[0114] SDS-PAGE has been used to analyze the expression of TrpS in biomass harvested from either 'Autoinduction in shake flasks' or 'Fermentation'. The SDS-PAGE samples were generally kept cold to avoid degradation of proteins.

[0115] Sampling: Culture volume corresponding to 2 OD units (2 / OD600) was aliquoted to a precooled 1.5 mL eppendorf tube on ice which was subsequently centrifuged for 2 min at 16000 x g at 4°C. The supernatant was removed with a pipette and the cell pellet was stored at -20°C until sample preparation. Sample preparation of the soluble fraction: The cell pellet was resuspended in 50 pL BugBuster Mastermix (room temperature) by pipetting. The suspension was incubated at 20°C 400 RPM (Eppendorf ThermoMixer C) for 15 min and then centrifuged at 16000 x g for 20 min. at 4°C.The supernatant (soluble fraction) was accurately transferred to a new pre-cooled eppendorf tube and stored at -20°C until sample dyeing. The pellet was used for sample preparation of the insoluble fraction. Sample preparation of the insoluble fraction: The pellet obtained from the sample preparation of the soluble fraction was resuspended in 50 pL BugBuster Mastermix (room temperature) by pipetting. 50 pL of 1:10 diluted BugBuster Mastermix (in ddH2O) was added to the suspension which was then vigorously vortexed. The suspension was centrifuged at 5000 x g for 15 min. at 4°C, and the supernatant was discarded by pipetting. The pellet (inclusion bodies) was resuspended in 50 pL of 1:10 diluted BugBuster Mastermix, mixed by vortexing, and centrifuged at 5000 x g for 15 min. At 4°C. The pellet was washed three times this way. Hereafter, the pellet wasonce more resuspended in 50 pL of 1:10 diluted BugBuster Mastermix and stored at -20°C until sample dyeing.

[0116] Sample dyeing: When the TrpS biomass was harvested from 'Autoinduction in shake flasks', the soluble fraction and the insoluble fraction were diluted 5 times (10.4 pL ddH2O + 2.6 pL protein sample). When the TrpS biomass was harvested from 'Fermentation', the soluble fraction was diluted 10 times (11.7 pL ddH2O + 1.3 pL protein sample) and the insoluble fraction was not diluted. 5 pL of 4x RunBlue LDS sample buffer and 2 pL of 500 pM DTT were added to 13 pL of proper diluted protein sample. To denature the proteins, the sample was incubated at 85°C for 3 min. The SDS-PAGE sample was stored at -20°C or loaded to an SDS-PAGE gel.

[0117] Gel loading and running: A 12% pre-cast gel (GenScript catalog number: M00669) was assembled into a Biorad Mini-Protean Tetra Gel Vertical Electrophoresis System which was used to run the SDS-PAGE. Freshly prepared lx MES running buffer (GenScript catalog number: M00677) was poured into the inner chamber, and no leakage must be observed. Then one time used or freshly prepared lx MES running buffer was poured into the outer chamber to the mark of 1-2 gels or 3-4 gels. The wells of the gel(s) were washed by pipetting 100 pL buffer from the inner chamber into each well a few times. 5 pL of protein marker (Precision Plus Protein Dual Color Standards from Biorad) was loaded to the first well and 10 pL of SDS-PAGE sample were carefully loaded to the other wells. The gel was run at 150 V for 75 min. Gel disassembling and staining: The gel chamber was disassembled, and the gel was placed in a tray containing ~25 mL RunBlue Instant blue staining dye. The gel must be completely covered by the stain. After ~1 h, the stain was poured into a waste container (special waste treatment needed), ddl-120 was poured into the tray with the gel, and after ~1 h it was exchanged with fresh ddl-120 for optimal de-staining. The gel was left in the water for at least one more hour or overnight (covered). Finally, the gel was pictured.3.4 BCA assay

[0118] The bioinchoninic acid (BCA) Protein Assay Kit from Novagen (product no. 71285) was used to quantify the amount of protein in various SDS-PAGE samples (protein samples). The assay exploits that proteins reduce Cu2+ to Cu+ in a basic solution, and the amount of protein is proportional to the amount of reduced Cu2+. The solution is green until 2 BCA molecules chelate with Cu+, and the solution turns purple on a scale reflecting the amount of protein.

[0119] The protocol for the micro-scale enhanced assay was followed using a 1:10 diluted BugBuster master mix (from SDS-PAGE sample preparation) as diluent. First, 6 bovine serum albumin (BSA) standards were prepared with concentrations ranging from 0 pg to 1000 pg. Protein samples of the soluble fraction were diluted 1:50 and protein samples of the insoluble fraction were diluted 1:10using a 1:10 diluted BugBuster Master Mix to be within the standard curve. The protein samples were kept cold on ice as much as possible. 25 pL of the BSA standards and protein samples were pipetted into a 96-well microtiter plate. The BCA working reagent was quickly prepared by mixing the provided 4% Cupric Sulfate solution and the provided BCA solution (bioinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 M NaOH, pH 11.25) in a ratio 1:50. 200 pL BCA working reagent was pipetted into each well with either BSA standard or protein sample. The content was mixed using a multichannel pipette, and a PCR seal was used as a lid. The 96-well microtiter plate was incubated at 60 °C for 15 min. and then cooled down to room temperature by standing for approximately 45 min. on the lab bench. The PCR seal was removed from the 96-well microtiter plate, and the absorbance was measured at 562 nm on a plate reader. The concentration of protein in each sample was calculated from the BSA standard curve.3.5 Biocatalysis

[0120] Biomass with TrpS was harvested from either Autoinduction in shake flasks or Fermentation. This biomass was used to perform TrpS biocatalysis converting L-serine to L-cysteine. The consumption of L-serine and production of L-cysteine and cystine were followed during the biocatalysis by taking samples for HPLC to assess the performance of TrpS. Cys_39 and Cys_309 were used as positive and negative control, respectively. It is established to use 50% biomass / g L-serine and a 1:1 molar ratio of L-serine and NaSH.

[0121] Preparation: Several solutions are needed to perform the TrpS biocatalysis. Stock solutions: 1) 1 M H2SO42) 10 mM H2SO43) 100 mM Tris-HCI buffer (pH 8.0) 4) Triton X-100. Solutions prepared on the day of the biocatalysis: 1) 600 g / L NaSH in MQ water 2) 70 g / L L-serine and0.1 mM PLP in 100 mM Tris-HCI buffer. An internal Cysbio Biocatalysis Excel Sheet was used to calculate the amounts needed which depends on how many biocatalysis reactors to run. 1221 pL NaSH solution and 10 mL Serine+PLP solution are the volume used for 1 reactor. When making the NaSH solution in a 50 mL falcon tube, the NaSH solution was shaken / vortexed and placed at 37°C for approximately 15 min. to get it dissolved.

[0122] Biocatalysis: 100 mL bluecap schott bottles were used as biocatalysis reactors. 10 mL Serine+PLP solution was added to each reactor, and 25.2 pL Triton X-100 was added subsequently. The Triton X-100 solution is very viscous making it difficult to pipette. Therefore, the pipetting should be done very slowly. Having the biocatalysis reactors ready, the harvested TrpS biomass was resuspended in 100 mM Tris-HCI buffer to 135 g wet weight / L and 2.59 m L of the resuspended biomasswas added to a reactor. When all the reactors had Serine+PLP, Triton X-100, and TrpS biomass, the biocatalysis was started immediately by adding 407 pL NaSH to one reactor, taking a TO sample (see HPLC sampling below), and then the reactor was incubated at 37°C 250 RPM (Biosan ES-20 / 80 Shaker- Incubator). A timer was started and the biocatalysis was started for one reactor at a time as fast as possible.

[0123] HPLC sampling and standards: Every hour, an HPLC sample was taken from one reactor at a time (in the same order) to keep the reactors in the incubator as much as possible during the biocatalysis. Importantly, the sampling was done in a fume hood. 407 pL NaSH was added to the reactor at every timepoint except at the last timepoint (end of biocatalysis). The content of the reactor was mixed by shaking carefully, and 100 pL from the reactor was pipetted to 100 pL 1 M H2SO4 (1:2 dilution) in the previously prepared 96-well plate. For 2-3 reactors at each timepoint, 10 pL was pipetted to a pH strip. When all samples were taken at a given timepoint, a multichannel pipette was used to mix the 1:2 diluted biocatalysis / acid sample and 40 pL was transferred to 1960 pL MQ water in the previously prepared 96-deep well plate resulting in a 100 times dilution. When all samples were taken for the biocatalysis, the 100 times diluted samples in the 96-deep well plate were mixed by pipetting, and 250 pL of each sample was filtrated through a 0.45 pM Hydrophilic Low Protein Binding Durapore Membrane into an HPLC plate. HPLC standards were prepared during and / or after the biocatalysis and 250 pL was added to the HPLC plate (no filtration needed). Similar L-serine and L- cysteine standards were prepared by making a 10 g / L stock solution (50 mg L-serine or L-cysteine to 5 mL 10 mM H2SO2) and diluting using 10 mM H2SO4 to make 1 g / L, 0.8 g / L, 0.6 g / L, 0.4 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 0.025 g / L, and 0.01 g / L standards. Cystine standards were made from a 50 g / L solution (50 mg cystine to 1 mLl M H2SO2) which was diluted 50 times using MQ water (500 pL 50 g / L cystine and 24.5 mL MQ water). The 1 g / L cystine stock solution and 10 mM H2SO2 was used to make 0.6 g / L, 0.4 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 0.025 g / L, and 0.01 g / L cystine standards.

[0124] HPLC: HPLC analysis was used to quantify L-serine, L-cysteine, and cystine using a Dionex UltiMate 3000 HPLC system from Thermo Scientific (LPG-3400RS Pump unit, WPS-3000 Autosampler unit, DAD-3000 Diode Array Detector unit and TCC-3000 Column Compartment unit) and a Primesep 100 column (150 x 4.6 mm with a 5 pm particle size and 100 °A pore size) equipped with a Primesep 100 guard column (4.6 x G mm, 5 pM, 100 °A) (SIELC Technologies). The mobile phases used were MilliQ water (A), 0.4% H2SO4 (B), and acetonitrile (C), and the applied flow gradient is specified in Table 9. The time for processing one HPLC sample was 12 min. The HPLC samples were kept at room temperature in the autosampler unit to avoid precipitation of L-cysteine and cystine, and the columnwas kept at 30° in the column compartment unit. Based on the peak areas of absorbance at 205 nm, L-serine, L-cysteine, and cystine were quantified in the freshly prepared standards and biocatalysis samples. The retention time for L-serine, L-cysteine, and cystine was 3.9 min, 4.3 min, and 6.4 min, respectively. Analysis was carried out using Chromeleon 7.2.10 (Thermo Fischer Scientific) software.Table 9: HPLC flow gradient for the quantification of L-serine, L-cysteine, and cystine.Example 4: Screening results of the mutants4.1 Soluble trpS mutants

[0125] As mentioned in example 1, 3 regions were identified as aggregation prone. 26 TrpS variants with mutation(s) in TrpB described in Table 1, designed for increased solubility of TrpB, were constructed and expressed.

[0126] The 26 TrpS variants, the current TrpS production strain (positive control, posC), and an identical strain with TrpS removed from the production plasmid (negative control, negC) were grown in batch fermentation conducted in shake flasks using autoinduction media to express TrpS. Samples of the soluble and insoluble fraction of proteins were prepared from 2 OD units of harvested biomass, and they were analyzed by SDS-PAGE and BCA assay. The SDS-PAGE results were used to qualitatively assess the expression of TrpA and TrpB in the soluble and insoluble fraction. The BCA assay was used to quantify the total amount of protein in the soluble and insoluble fraction. Having qualitative and quantitative data supporting each other (Figure 8, Figure 9, and Figure 10), it was possible to identify TrpS variants (V227P, C230E, V231D, V276K, V276E, G277D, I278E, Y279K, F280E, Q370E, N375P) which have reduced expression of TrpB in the insoluble fraction compared to the positive control.4.2 TrpS variants stabilizing the amino acrylate conformation

[0127] As mentioned in example 1, 316 mutations were identified to stabilize the closed E(A-A) conformation of TrpB. Among the mutations predicted to stabilize the E(A-A) conformation, S163R and N246Y were engineered and tested. The biomass used to perform biocatalysis was produced in batch fermentation conducted in shake flasks using autoinduction media. The current TrpS production strain was included as positive control (+ve), and the same strain with TrpS removed from the production plasmid was included as negative control (-ve). The result of the biocatalysis screening isshown in Figure 11. S163R and N246Y are active. To test if the active TrpS variants also displayed increased catalytic activity in conditions resembling large-scale production conditions, S163R and N246Y were produced by fed-batch fermentation, and the biocatalytic activity were compared to the currently used TrpS enzyme (+ve). The result of the biocatalysis using biomass from fed-batch fermentation is shown in Figure 12. The total productivity (produced cysteine+cystine in mmol / h) of both S163R and N246Y are increased compared to the positive control (+ve). Hence, the mutateX analysis described in example 1 can be used to identify mutations that stabilizes the E(A-A) conformation of TrpB resulting in more active TrpS variants.

[0128] S163R is one example of a structural explanation on how the E(A-A) conformation of TrpB can be stabilized. An energy-minimized model of the wild-type and S163R TrpB structures was examined both in the E(Aexl) and the E(A-A) conformation (Figure 13). The wild-type structure of TrpB reflects the currently used TrpS enzyme at industrial scale (positive control). In the E(Aexl) conformation, the side-chain of S163 interacts with T165, K167, and D168 in the COMM domain through hydrogen bonding, and the main-chain interacts with K137 and D168 in the COMM domain through hydrogen bonding (Figure 13A). The distance between K167 in the COMM domain and E296 in the long loop is 6.2 A indicating that there is no salt bridge formation in the E(Aexl) conformation of TrpB. In contrast, the distance between K167 in the COMM domain and E296 in the long loop in the E(A-A) conformation of TrpB is only 4.3 A which indicates that a salt bridge has been formed (Figure 13B). This results in the loss of a hydrogen bond between K167 and the side-chain of S163 in the E(A-A) conformation of TrpB. The conformational change from E(Aexl) to E(A-A) also causes the loss of a hydrogen bond between the main-chain of S163 and K137. The observations for the wild type TrpS S163 in the E(Aexl) and E(A- A) conformation can be compared to the TrpS mutant R163. In the E(Aexl) conformation, the sidechain of R163 forms a salt bridge to E296 (distance is 2.6 A) in the long loop (Figure 13C). It is likely that this salt bridge compensate for the lost hydrogen bonds which was observed for S163. The distance between K167 and E296 is 6.1 A which is similar to 6.2 A in the S163 E(Aexl) conformation. In the E(A-A) conformation, the side-chain of R163 in the COMM domain forms several salt bridges to D305 and several hydrogen bonds to S297 in the long loop which is not observed for S163 in the E(A- A) conformation (Figure 13D). At the same time, the salt bridge between K167 and E296 is still observed (distance is 4.2 A), and R163 is also forming a hydrogen bond to T165 in the COMM domain as observed for S163. Hence, mutating S163 to arginine results in a net loss of 2 interactions in the E(Aexl) conformation, but the interactions lost are hydrogen bonds whereas the interaction formed is a salt bridge, which has substantially higher bonding energy, explaining why E(Aexl) is not destabilized even though there is a net loss of interactions. In the E(A-A) conformation, mutating S163to arginine results in the formation of 5 new interactions suggesting that E(A-A) is significantly stabilized. This is exactly what we aimed for in the design process using MutateX which calculated the E(Aexl) AAG to be -0.08 kcal / mol and the E(A-A) AAG to be -3.92 kcal / mol (Table 2). Considering the accuracy of the MutateX design approach, it is possible that it can be used to identify mutations which stabilize one conformation more than another conformation in other protein engineering studies. In addition to the influence of the S163R mutation on stability, it is also important to consider how the mutation affects the functions of key residues. As previously described, it has been reported that a salt bridge between K167 and E296 is important for the stabilization of TrpB and more specifically for the stabilization of the long loop (Nishio, et al., 2010). Therefore, it is essential that this salt bridge is still able to form when the mutation S163R is introduced in TrpB. K167 is an important residue because it is in the TrpB interface (within 3 A of TrpA), and it is involved in the signal transmission between the subunits (Raboni, Bettati, & Mozzarelli, 2009) (Nishio, et al., 2010). Importantly, S163R does not interfere with K167 either. D305 has been reported to have several functions such as forming salt bridges to other residues and hence stabilizing the long loop, and it is also a part of the active site where it interacts with serine through hydrogen bonding (Nishio, et al., 2010) (Rhee, et al., 1997). Even though R163 forms salt bridges with D305, it is possible that this does not disrupt other important interactions of D305 because the conformation stays the same. From a structural point of view, the mutation S163R stabilizes the E(A-A) conformation by increasing the interactions between the COMM domain and the long loop, without negatively affecting the E(Aexl) conformation or any of the key functional residues important for the catalytic activity of TrpB. Stabilizing the E(A-A) conformation of TrpB by introducing mutations that increases the interactions between the COMM domain (residue 102-189:GKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRD WSGSYETAHYMLG) and the long loop (residue 260-310: HGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSISAGLDFPSVG) are a general engineering approach that can be used to increase the activity of TrpS.4.3 TrpS variants stabilizing the closed conformation of TrpA

[0129] As mentioned in example 1, 7 mutations were identified to lock TrpA in its closed conformation which inturn promotes TrpB to get in to its closed conformation faster. The disulfide bridges were used to make the closed conformation. 2 TrpS variants (S178C_F212C, R179C_G189C) were engineered and tested via biocatalysis using biomass from batch fermentation conducted in shake flasks using autoinduction media. Kindly note that the TrpA protein is N terminally His tagged for easy purification of protein is required, However, the amino acid residue numbers corresponds towild type TrpA. The result of the biocatalysis screening is shown in Figure 14. S178C_F212C and R179C_G189C produce cysteine at higher rates compared to the positive control (+ve). To test if the active TrpS variants displayed increased catalytic activity in conditions resembling large-scale production conditions, S178C_F212C and R179_G189C were produced by fed-batch fermentation, and the biocatalytic activity were compared to the currently used TrpS enzyme (Figure 15). The total productivity (produced cysteine+cystine in mmol / h) of both S178C_F212C and R179_G189C are increased compared to the positive control (+ve). Hence, locking TrpA in its closed conformation can increase the enzymatic activity of TrpS.

[0130] A structural investigation of R179C_G189C in an energy-minimized model of the closed conformation of TrpA is shown in Figure 16. The wild-type structure of TrpA reflects the currently used TrpS enzyme at industrial scale (positive control). It is notable that R179 interacts with S180, V182, G184, and N187 through hydrogen bonding. These interactions suggest that R179 is important for stabilizing Loop6 in the closed conformation of TrpA. G189 is not involved in hydrogen bonding, and it does not play a role in the closing of Loop6. R179 and G189 were mutated to cysteine, and it is confirmed in an energy-minimized model that they can form a disulfide bond under oxidizing conditions. The disulfide bond stabilizes Loop6 and keeps TrpA in its closed conformation. Locking TrpA in its closed conformation by engineering of Loop6 (residue 178-192: SRAGVTGAENRAALP) can increase the activity of TrpS.Example 5: Construction of Fusion TrpAB construct.

[0131] TrpS is dimer of dimer, which means 4 proteins have to align in right orientation to make an active construct. We hypothesized that if we can make a fusion construct we can remove the requirement to just dimer structure. Such a construct exists natively in yeast (Zalkin & Yanofsky, 1982). First the TrpA-TrpB construct was made (Example 5.1). For increasing flexibility of TrpA and TrpB for best alignment to each other we constructed linker sequences of amino acid length varying from 5 amino acid to 16 amino acid (Example 5.2). The TrpAB fusion constructs with various linker lengths have SEQ ID NO: 367 to 378, and the various linkers have SEQ ID NO: 379 to 390.5.1 Construction of TrpAB fusion construct

[0132] For constructing the TrpAB fusion construct the position of TrpA and TrpB were swapped using PCR products amplified by primers mentioned in Table 10.Table 10: Primers used to make the TrpAB fusion construct.The key variables from the PCR details mentioned in example 2 are listed in Table 11.Table 11: PCR details for amplification of TrpAB fusion construct

[0133] The fragments of right size were obtained and were colum purified after Dpnl digestion the respective biobricks number are given in above table.

[0134] 0.5 ul each of the above fragments were added to the Gibson reaction. The rest of the cloning procedure to obtain the plasmid is as mentioned in example 2. The resultant plasmid constructed is pCys_125 with Linker 10AA.5.2 Construction of trpAB fusion construct with variable length

[0135] To construct the TrpAB fusion construct with variable length, the plasmid pCys_125 was used as a template to extend linker length. The primers given in Table 12 were ordered.Table 12: Primers to extend the linker length

[0136] The key variables for each PCR amplification are given in Table 13. The overall protocol is summarized in example 2.Table 13: PCR amp ification of TrpAB fusion construct with variable length

[0137] 50 pL PCR product was treated with Dpnl for 1 h at 37°C and then column purified. 5' Phosphate was added using Poly nucleotide kinase reaction and then subjected for intermolecular ligation using T4 DNA ligase before transformation. The details for plasmid isolation and retransformation in production host are given in example 2. The resultant production strains were tested for expression studies and biocatalysis. All TrpS variants were expressed in shake flask experiment. Interestingly, all of them were expressed only in inclusion body (example 6). Nevertheless, biomass was tested for catalysis and surprisingly the variants with linker lengths showed best activity (example 6).Example 6 Study of TrpS variants with varying lengths

[0138] The biocatalysis data from shake flask (Figure 17) and SDS-PAGE analysis (Figure 18 and Figure19) from key strains subjected for fermentation show that inspite being completely in insoluble fraction, all selected TrpAB fusion constructs are active. Thus, proving they are catalytically active inclusion bodies.Example 7: Reuse of TrpS

[0139] It was investigated if the insoluble TrpAB fusion construct could be reused in biocatalysis because this has significant potential at industrial scale. Biocatalysis reusing the soluble and insoluble fraction of the currently used TrpS (Cys_39) and one of the TrpAB fusion constructs (Cys_197) was performed, and the results are shown in Figure 20. TrpS (Cys_39) loses its activity when reused whereas the insoluble TrpAB fusion construct (Cys_197) remain active. As shown in Figure 21, the recovery rate (%) of the insoluble TrpAB fusion construct (Cys_197) after 20 min of biocatalysis is 87.8% from round 1 to round 2, 83.7% from round 2 to round 3, and 73.5% from round 1 to round 3. The decrease in recovery rate (%) is expected to be mainly because of experimental loss of enzyme between the rounds. Hence, it has been proved that the insoluble TrpAB fusion construct can be reused in biocatalysis.Biocatalysis reusing TrpAB fusion construct biomass was performed, and the results are shown in Figure 22. The TrpAB fusion construct is active for 5 rounds of biocatalysis even though the TrpAB fusion construct biomass has been stored at 4°C overnight. These results prove that the TrpAB fusion construct is highly stable, and it can be used in a continuous biocatalysis process as the TrpAB fusion construct seems to be active for many rounds of biocatalysis. From a process point of view, it is convenient that no enzyme isolation or purification is required as the TrpAB fusion construct biomass can be used to perform biocatalysis.ReferencesAshkenazy, H., Abadi, S., Martz, E., Chay, O., Mayrose, I., Pupko, T., & Ben-Tai, N. (2016). ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic acids research, pp. W344-W350.Esaki, N., Tanaka, H., Miles, E. W., & Soda, K. (1983). Enzymatic synthesis of S-substituted L-cysteines with tryptophan synthase of Escherichia coli. Agricultural and biological chemistry, pp. 2861- -2864.Fernandez-Escamilla, A.-M., Rousseau, F., Schymkowitz, J., & Serrano, L. (2004). Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nature biotechnology, pp. 1302-1306.Hyde, C., Ahmed, S., Padlan, E., Miles, E. W., & Davies, D. (1988). Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. Journal of Biological Chemistry, pp. 17857-17871.Ishiwata, K.-i., Nakamura, T., Shimada, M., & Makiguchi, N. (1989). Enzymatic production of L- cysteine with tryptophan synthase of Escherichia coli. Journal offermentation and bioengineering, pp. 169-172.Ismail, N. I., Hashim, Y. Z.-Y., Jamal, P., Othman, R., & Salleh, H. M. (2014). Production of cysteine: approaches, challenges and potential solution. International Journal of Biotechnology for Wellness Industries, pp. 95-101.Nishio, K., Ogasahara, K., Morimoto, Y., Tsukihara, T., Lee, S. J., & Yutani, K. (2010). Large conformational changes in the Escherichia coli tryptophan synthase beta2 subunit upon pyridoxal 5'-phosphate binding. The FEBS Journal, pp. 2157-2170.Noske, J., Kynast, J. P., Lemm, D., Schmidt, S., & Hocker, B. (2023). PocketOptimizer 2.0: A modular framework for computer-aided ligand-binding design. Protein Science.Raboni, S., Bettati, S., & Mozzarelli, A. (2009). Tryptophan synthase: a mine for enzymologists. Cellular and molecular life sciences, pp. 2391-2403.Rhee, S., Parris, K., Hyde, C., Ahmed, S., Miles, E., & Davies, D. (1997). Crystal Structures of a Mutant (betaK87T) Tryptophan Synthase alpha2beta2 Complex with Ligands Bound to the Active Sites of the alpha- and beta-Subunits Reveal Ligand-Induced Conformational Changes. BIOCHEMISTRY-PENNSYLVANIA THEN WASHINGTON-, pp. 7664-7680.Schymkowitz, J., Borg, J., Stricher, F., Nys, R., Rousseau, F., & Serrano, L. (2005). The FoldX web server: an online force field. Nucleic acids research, pp. W382-W388.Swift, S., & Stewart, G. S. (1991). The molecular biology of tryptophan synthase: A model for proteinprotein interaction. Biotechnology and Genetic Engineering Reviews, 229-294.Tiberti, M., Terkelsen, T., Degn, K., Beltrame, L., Cremers, T. C., da Piedade, I., . . . Papaleo, E. (2022). MutateX: an automated pipeline for in silico saturation mutagenesis of protein structures and structural ensembles. Briefings in bioinformatics.Van Durme, J., De Baets, G., Van Der Kant, R., Ramakers, M., Ganesan, A., Wilkinson, H., . . . Schymkowitz, J. (2016). Solubis: a webserver to reduce protein aggregation through mutation. Protein Engineering, Design and Selection, pp. 285-289.Watkins-Dulaney, E., Straathof, S., & Arnold, F. (2021). Tryptophan synthase: biocatalyst extraordinaire. ChemBioChem, pp. 5-16.Wilcox, M. (1974). The enzymatic synthesis of l-tryptophan analogues. Analytical Biochemistry, pp. 436-440.Zalkin, H., & Yanofsky, C. (1982). Yeast gene TRP5: structure, function, regulation. Journal of Biological Chemistry, pp. 1491-1500.* * *

Claims

Claims1. A mutant tryptophan synthase (TrpS) comprising two moieties, a and p, having amino acid sequences which are at least 70% identity to amino acid moieties SEQ ID NO: 1 (TrpA) and 2 (TrpB) respectively, and comprising at least one mutation selected from: a) a substitution of an amino acid in a and / or ; b) a deletion of an amino acid in a and / or P; c) an insertion of an amino acid in a and / or P; and / or d) a fusion of a and P; compared to a parent TrpS comprising a and p moieties having amino acid sequences corresponding to SEQ ID NO: 1 and 2.

2. The mutant TrpS of claim 1 having increased specific activity and / or increased or reduced water solubility compared to the parent TrpS.

3. The mutant TrpS of any preceding claim wherein the mutation promotes a fold type II protein conformation and demotes a fold type I protein conformation protein conformation.

4. The mutant TrpS of any preceding claim wherein the mutation increases stability of a E(A-A) conformation of the p moiety by introducing mutation in: a) a COMM domain of the p moiety corresponding to residue 102-189 of SEQ ID NO: 2, specified as SEQ ID NO: 872, and / or b) a long loop domain of the p moiety corresponding to residue 260-310 of SEQ ID NO: 2, specified as SEQ ID NO: 874, which mutation increases interactions between the COMM domain and the long loop domain and thereby increases the activity of the mutant TrpS compared to the unmutated TrpS.

5. The mutant TrpS of any of claims 1 to 3 wherein the mutation increases or locks a closed conformation of the a moiety by introducing mutation in a Loop6 domain of the a moiety corresponding to residue 178-192 of SEQ ID NO: 1, specified as SEQ ID NO: 870, and thereby increases the activity of the mutant TrpS compared to the unmutated TrpS.

6. The mutant TrpS of any preceding claims wherein the substitution in the p moiety corresponds to mutations in SEQ ID NO: 2 selected from S163R (SEQ ID NO: 52), N246Y (SEQ ID NO: 47).

7. The mutant TrpS of any preceding claims wherein the substitution in the a moiety corresponds to mutations in SEQ ID NO: 1 selected from S178C+F212C (SEQ ID NO: 5) and / or R179C+G189C (SEQ ID NO: 6).

8. The mutant TrpS of any of claims 1 to 3 wherein the a and p moieties are fused into a fusion construct.

9. The mutant TrpS of claim 8 wherein the fusion construct comprising a linker sequence between a and moieties wherein the a moiety becomes before the p moiety.

10. The mutant TrpS of any of claims 8 to 9 wherein the linker sequence has a sequence which is at least 70% identical to the linker comprised in any of SEQ ID NO: 379 to 390.

11. The mutant TrpS of any of claims 8 to 10 wherein the fusion construct has an amino acid sequence which is at least 70% identical to the fusion construct comprised in anyone of SEQ ID NO: 367 to 378.

12. The mutant TrpS of claim 11 wherein the fusion construct is less soluble in aqueous solution than the unfused a and p moieties.

13. The mutant TrpS of claim 12 wherein the fusion construct is insoluble in aqueous solution.

14. The mutant TrpS of claim 8 to 13 wherein the fusion construct is catalytically active in aqueous solution, and optionally re-used in unlimited numbers of biocatalysis.

15. The mutant TrpS of any preceding claim being a heterotetramer comprising appa in a heterotetrameric structure.

16. A genetically modified host cell expressing or overexpressing the mutant TrpS or a or p or linker moieties of claims 1 to 15.

17. The host cell of claim 16 wherein the host cell is a prokaryotic cell, optionally a bacterium.

18. The host cell of claim 17 wherein the prokaryotic cell is a cell of Pseudomonadota, optionally of the class gammaproteobacteria, optionally of the family Enterobacteriaceae, optionally of the genusEscherichia, optionally of the species Escherichia coli.

19. The host cell of claim 17 wherein the prokaryotic cell is an Actinomycetota, optionally of the class Actinobacteria, optionally of the family Corynebacteriaceae, optionally of the genus Corynebacterium, optionally of the species Corynebacterium glutamicum.

20. A method for producing cysteine or cystine comprising contacting L-serine with the TrpS mutant of any of claims 1 to 15 in an aqueous medium in the presence of a source of sulfur to produce the cysteine or cystine; and optionally recovering and / or isolating cysteine or cystine.* * *

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